Hepatitis C virus (HCV) infects 3% of the world population and accounts for most cases of chronic liver disease. In the United States, HCV infection is the leading cause of liver failure. Drugs targeting HCV proteins are difficult to be developed owing to the high rate of mutation during HCV replication that allows quick appearance of the drug-resistant viral strains. The current treatment for HCV infection is based on interferon, which mediates an innate immune response against infection of RNA virus. However, this treatment is only partially effective. Thus, understanding other innate antiviral responses may reveal much needed new strategies to treat HCV infection. We have recently identified a novel innate antiviral response that plays an important role in limiting HCV infection in a line of human hepatoma cells. This pathway is mediated by cAMP response element binding protein 3-like 1 (CREB3L1), the function of which was previously unknown. CREB3L1 belongs to a family of transcription factors that are synthesized as membrane-bound precursors inserted in the endoplasmic reticulum (ER), and activated by a process termed regulated intramembrane proteolysis (RIP). Based on our current understanding of RIP, we propose that replication of HCV in the ER results in cleavage of CREB3L1 by Site-1 protease (S1P) and Site-2 protease (S2P). The proteolytic cleavage allows the NH2-terminal fragment of CREB3L1 to be released from the membrane and travel to the nucleus to activate its target genes involved in antiviral responses. These hypotheses will be tested by three specific aims raised in the proposal. Specific Aim 1 will determine the mechanism by which HCV replication stimulates the cleavage of CREB3L1. We will examine whether ER stress induced by expression of HCV- encoded membrane proteins triggers the cleavage of CREB3L1. Specific Aim 2 will determine whether S1P and S2P-catalyzed cleavages of CREB3L1 is required for its antiviral function. The primary approach is to make CREB3L1 mutants that cannot be cleaved by these proteases and examine the effect of the mutations on its antiviral function. Specific Aim 3 will identify the CREB3L1 target genes that inhibit HCV replication by microarray analysis. If these specific aims are achieved, we will have contributed novel information that will significantly enhance our understanding of the innate immune response. This new knowledge may reveal novel drug targets to treat HCV infection.